In order to develop biocatalytic methods for the manufacture of pure single enantiomer compounds, effective procedures must be found to discover enzymes that are highly enantioselective and also enzymes that can provide any desired stereoisomer of a given chiral compound. Identification of highly stereoselective enzymes from a large clonal library is a very challenging task.
Transaminases have been used for the synthesis of natural amino acids for some time. More recently attention has turned to the use of transaminases for the synthesis of chiral unnatural amino acids and chiral amines as illustrated in FIGS. 1A and 1B. Transaminases are a group of key enzymes in the metabolism of amino acids and amino sugars and are found in all organisms from microbes to mammals. In the transamination reaction, an amino group is transferred from an amino acid to an alpha-keto acid. Pyridoxal phosphate is frequently required as a co-factor to mediate the transfer of the amino group without liberation of ammonia. Two important types of organic chiral precursors are chiral unnatural amino acids and chiral amines. Chiral unnatural amino acids and chiral amines can be conveniently produced through catalysis of transaminases as illustrated herein.
There are several attractive features of transaminases that make them promising catalysts for large-scale production of chiral amino acids and amines. These include: (1) No cofactor regeneration is required. Transaminases do not require nicotinamide cofactors. Instead the cofactor (pyridoxal phosphate) is tightly bound to the enzyme. (2) Transaminases are generally highly enantioselective.
One of the main limitations to the widespread application of transaminases is the lack of available enzymes. For example, the current enzymes accept only a limited number of substrates and often suffer from product inhibition. Transaminase reactions are reversible and the equilibrium constant is generally near unity. This can be a challenge in obtaining high yields of products.
Transaminase-catalyzed syntheses of chiral amines and amino acids can be limited by the equilibrium of the reaction which is generally close to unity. Several approaches to driving the equilibrium in the direction of product formation have been described. For example, (1) Addition of molar excesses of amino donors; (2) Removal of alpha-keto acid product either enzymatically or chemically, (3) If the value of the target amine is high enough, it may not be necessary to drive the equilibrium to completion.
There is a need in the chemical industry for efficient catalysts for the practical synthesis of optically pure materials. Enzymes can provide the optimal solution. All classes of molecules and compounds that are utilized in both established and emerging chemical, pharmaceutical, textile, food, animal feed, and detergent markets must meet stringent economical and environmental standards. The synthesis of polymers, pharmaceuticals, natural products and agrochemicals is often hampered by expensive processes, which produce harmful byproducts and which suffer from low enantioselectivity.
Enzymes have a number of remarkable advantages, which can overcome these problems in catalysis: they act on single functional groups, they distinguish between similar functional groups on a single molecule, and they distinguish between enantiomers. Moreover, they are biodegradable and function at very low concentrations in reaction mixtures. Because of their chemo-, regio- and stereospecificity, enzymes present a unique opportunity to optimally achieve desired selective transformations. These are often extremely difficult to duplicate chemically, especially in single-step reactions. The elimination of the need for protection groups, the selectivity of enzymes, the ability to carry out multi-step transformations in a single reaction vessel, along with the concomitant reduction in environmental burden, has led to the increased demand for enzymes in chemical and pharmaceutical industries. Enzyme-based processes have been gradually replacing many conventional chemical-based methods. A current limitation to more widespread industrial use is primarily due to the relatively small number of commercially available enzymes.
The use of enzymes for technological applications may require performance under demanding industrial conditions. This includes activities in environments or on substrates for which the currently known arsenal of enzymes was not evolutionarily selected. Enzymes have evolved by selective pressure to perform very specific biological functions within the milieu of a living organism, under conditions of mild temperature, pH and salt concentration. For the most part, the non-DNA modifying enzyme activities thus far described have been isolated from mesophilic organisms, which represent a very small fraction of the available phylogenetic diversity. The dynamic field of biocatalysis takes on a new dimension with the help of enzymes isolated from microorganisms that thrive in extreme environments. Enzymes obtained from these extremophilic organisms open a new field in biocatalysis.
Transaminases have been known in the literature for many years. Briefly, a transaminase reaction requires two substrates, an amino compound (amino donor) and a keto compound (amino acceptor). The transaminase catalyzes the exchange of the keto group from the keto compound and the amino group from the amino compound. This exchange generates a new amino compound from the keto compound and a new keto compound from the amino compound. Typically only one of the products is desired, generally the new amino compound, and the other is an unwanted by-product. Used in isolation, the enzyme converts the two substrates to the two products. Theoretically, because the reaction is reversible, it proceeds until it reaches equilibrium.
U.S. Pat. No. 4,518,692 (“Rozzell I”) discloses a method for producing L-amino acids by reacting L-aspartic acid and various 2-keto acids with transaminases. The Rozzell I method uses L-aspartic acid as the amino acid to produce oxaloacetate and describes various methods of decarboxylating oxaloacetate to form pyruvate. However, the pyruvate produced in the Rozzell I method can still act as a keto donor in the reverse process to form alanine. Tokarski et al., Biotechnology Letters, Vol. 10 (1) (1988), pp. 7-10, show that alanine acts as a substrate in transaminase reactions. See also, Transaminases (1985); and Amino Acids: Biosynthesis and Genetic Regulation, Klaus M. Herrmann and Ronald L. Somerville ed. (1983) (Addison-Wesley Publishing, Reading Mass.). Tokarski, et al. studied the use of a transaminase to produce L-2-aminobutyrate from 2-ketobutyrate and alanine. The reference, however, discloses only 25-30% conversion to products, demonstrating that the reverse reaction is very significant. This has long been considered an intrinsic property and a problem of transaminase reactions and is the major reason such enzyme catalyzed reactions have not been more often exploited in industrial processes to produce these highly desired amine products.
U.S. Pat. No. 4,826,766 (“Rozzell II”) discloses an improved transaminase catalyzed reaction that employs two transaminase enzymes and additional keto acids. In the process, a first transaminase enzyme catalyzes the reaction between a first amino acid and a first keto acid to produce a second amino acid and second keto acid. A second transaminase enzyme then catalyzes a further reaction of the second amino acid and a third keto acid to form the desired amino acid. The two transaminase enzymes are selected such that the first enzyme does not catalyze the second reaction and the second enzyme does not catalyze the first reaction.
Another transaminase process, which combines the transaminase enzyme with a second enzyme that eliminates the keto acid produced by the transaminase reaction, preventing the attainment of equilibrium, and driving the amino acid producing reaction to completion, is known from U.S. Pat. No. 6,197,558 (Fotheringham). The second enzyme catalyzes a reaction, which converts the keto acid to a substance that can no longer react with the transaminase. By removing the second keto acid, the second enzyme allows the amino acid producing reaction to proceed to an extent that the desired amino acid product represents approximately 100% of the amino acids produced.
Aldolases are ubiquitous enzymes that catalyze the formation of carbon-carbon bonds through the aldol reaction (FIG. 7). Depending on the donors and acceptors utilized, the reaction generates one or two new stereocenters (indicated by the asterixes in FIG. 1). Thus, aldolases have great potential for the production of advanced chiral products that are difficult and/or expensive to produce by traditional chemical routes. FIG. 8 illustrates a few well-characterized examples of reactions catalyzed by aldolases. In regard to substrate specificity, the aldehyde acceptor component can be varied to some extent (FIG. 3), and the enolate donor requirement is typically quite strict. Some examples of aldolase-mediated synthesis with non-natural substrates have been reported, although these cases are currently limited (see, e.g., JOC 2000, 95, 8264; b. JACS 1996, 118, 2117; c. JACS 1997, 119, 11734). Realization of the synthetic potential of aldolases in large scale industrial processes has been limited by the lack of available enzymes with the necessary properties.
There are two major routes from a nitrile to an analogous acid: (1) a nitrilase catalyzes the direct hydrolysis of a nitrile to a carboxylic acid with the concomitant release of ammonia; or (2) a nitrile hydratase adds a molecule of water across the carbon-nitrogen bonding system to give the corresponding amide, which then acts as a substrate for an amidase enzyme which hydrolyzes the carbon-nitrogen bond to give the carboxylic acid product with the concomitant release of ammonia. The nitrilase enzyme therefore provides the more direct route to the acid.
A nitrile group offers many advantages in devising synthetic routes in that it is often easily introduced into a molecular structure and can be carried through many processes as a masked acid or amide group. This is only of use, however, if the nitrile can be unmasked at the relevant step in the synthesis. Cyanide represents a widely applicable C1-synthon (cyanide is one of the few water-stable carbanions) which can be employed for the synthesis of a carbon framework. However, further transformations of the nitrile thus obtained are impeded due to the harsh reaction conditions required for its hydrolysis using normal chemical synthesis procedures. The use of enzymes to catalyze the reactions of nitrites is attractive because nitrilase enzymes are able to effect reactions with fewer environmentally hazardous reagents and by-products than in many traditional chemical methods. Indeed, the chemoselective biocatalytic hydrolysis of nitrites represents a valuable alternative because it occurs at ambient temperature and near physiological pH.
The importance of asymmetric organic synthesis in drug design and discovery has fueled the search for new synthetic methods and chiral precursors which can be utilized in developing complex molecules of biological interest. One important class of chiral molecules are the α-substituted carboxylic acids, which include the α-amino acids. These molecules have long been recognized as important chiral precursors to a wide variety of complex biologically active molecules, and a great deal of research effort has been dedicated to the development of methods for the synthesis of enantiomerically pure α-amino acids and chiral medicines.
Of particular use to synthetic chemists who make chiral medicines would be an enzyme system which is useful under non-sterile conditions, which is useful in non-biological laboratories, which is available in a form convenient for storage and use; which has broad substrate specificity, which acts on poorly water soluble substrates; which has predictable product structure; which provides a choice of acid or amide product; and which is capable of chiral differentiation. Accordingly, there is a need for efficient, inexpensive, high-yield synthetic methods for producing enantiomerically pure α-substituted carboxylic acids, such as, for example, α-amino acids and α-hydroxy acids.
Chiral epoxides and diols are key building blocks for the synthesis of pharmaceuticals. The epoxide group is readily transformed into a wide range of derivatives by acid or base-catalyzed ring opening reactions, while the diols similarly can be converted into a diverse range of structures. Currently available methods for the enantioselective preparation of chiral epoxides and diols have drawbacks that limit their use in industrial applications. Epoxide hydrolases (E.C. 3.3.2.x) are attractive as biocatalysts for the preparation of chiral epoxides and vicinal diol as they can selectively hydrolyze one of the enantiomers. The selective hydrolysis of a racemic epoxide thus generates both the corresponding diols and the unreacted epoxides with high enantiomeric excess (ee) values.